To appreciate how cells migrate, establish polarity, and adopt cell shape is fundamental to understanding cancer biology. Almost one percent of the human proteome is dedicated to Rho GTPase signaling, which regulates key aspects of each of these processes. It is well known that Rho GTPases are "signaling switches" that are turned "on" or "off" by GEF (Guanine nucleotide Exchange Factors) and GAP (GTPase Activating Proteins) proteins. What is not known are the molecular mechanisms that determine Rho GEF and GAP specificity. Because GEFs and GAPs regulate Rho-GTPases in specific cellular contexts, they are promising candidates for future therapeutic interventions to modulate GTPase activity in different types of cancers. This proposal develops an innovative and integrated approach to map the architecture of Rho GTPase signaling in vivo. Multiple independent methods will be used, including novel proteomic screens using cellular proteins with unnatural amino acids as well as high throughput binding assays and informatics- based experiments. We have recently established this approach in our laboratory and our preliminary data validates our rationale that this approach will uncover novel links between Rho GTPase signaling and cancer biology. At the conclusion of this pilot project we expect to demonstrate that this approach can work on a larger scale to map interaction networks for regulators of this pathway. These results will fundamentally advance our understanding of how Rho GTPases are modulated to regulate processes such as migration, polarity, and cell morphology. It can also be expected to provide the basic information necessary for the future development of therapeutic strategies to alter cancer outcomes.